Figure 4.

Pharmacological inhibition of Nox4 enzymatic activity prevents hyperproliferation by H-RasV12. (a) Nox4i treatment reduces ROS accumulation in RasV12 cells as detected by the DHE probe. Scale bar: 40 μm. (See also Supplementary Figure S4A). (b) Two distinct NOX4 inhibitors (NOX4i1, NOX4i2), but not their vehicle (DMSO) prevent RasV12-induced hyperproliferation and OIS establishment. (c) BrdU incorporation rates measured at the indicated time points of the growth curve displayed in panel a show that both hyperproliferation and cellular senescence are abolished by Nox4i treatment. Differences in the rates of BrdU incorporation between RasV12-expressing cells treated by DMSO versus the ones treated by either Nox4i1 or Nox4i2 inhibitors at day 5 (the peak of the hyperproliferative phase) are statistically significant (*P-value < 0.01). (d) Immunostaining of cells at the last time point of the growth curves shown in b indicates that DDR activation is strongly reduced by Nox4i treatment. The percentages of DDR in the form of 53BP1 and γH2AX foci of cells are compared individually among RasV12-expressing cells with respect to treatment using two different Nox4 inhibitors. Error bars indicate S.E.M. (n≥ 3), and differences are statistically significant (*P-value < 0.01) throughout the figure where stated